Transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) we... more Transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) were subjected to two methods of extraction: (1) freezing/thawing/sonication (FTS) cycles prior to the three-phase partitioning (TPP) method, or (2) directly to TPP extraction. The amount of GFPuv released by the FTS plus TPP method varied: 374µg/mL (first cycle), 93–442 µg/mL (second cycle), 32–359 µg/mL (third cycle), 18–115 µg/mL (fourth cycle). The GFPuv yields by the second method (TPP only) were, 23–54 µg/mL for the first extract and 33–91 µg/mL for the second. The FTS plus TPP method released similar amounts of GFPuv to that extracted by TPP; and provided a better mixture elution through the hydrophobic interaction column: 13–63 µg/mL for FTS plus TPP methods, and 2.5–13 µg/mL for TPP. The results showed that although selective permeation is a more laborious methodology, it was more efficient for obtaining of GFPuv in relation to the direct extraction of the cells for TPP.
Brazilian Archives of Biology and Technology, 2003
From analytical to commercial scale, aqueous two-phase systems have their application in the puri... more From analytical to commercial scale, aqueous two-phase systems have their application in the purification, characterization and study of biomaterials. In order to improve the selectivity of the systems, the biospecific affinity ligands were introduced. In the affinity partitioning aqueous two-phase system, have many enzymes been purified. This review discusses the partitioning of some enzymes in the affinity aqueous two-phase systems in regard to the different ligands, including reactive dyes, metal ions and other ligands. Some integration of aqueous two-phase system with other techniques for more effective purification of enzymes are also presented.
Bioprocess, Bioseparation, and Cell Technology, 2009
ABSTRACT The study of biomolecules and bioparticles has achieved great advances, thus increasing ... more ABSTRACT The study of biomolecules and bioparticles has achieved great advances, thus increasing the productivity with upstream improvements. Nowadays, downstream processes are the bottleneck in the production of some biopharmaceuticals, a change from the previous years' scene. Current purification platforms will reach their physical limits at some point, creating the necessity for new approaches. This article reviews an alternative method for the extraction and purification of biomolecules/bioparticles denominated as aqueous two-phase systems (ATPSs). Biocompatibility and readiness to scale up are some of the characteristics of ATPSs and we also discuss its application in the biotechnology field.Keywords:polymer/polymer aqueous two-phase systems;polymer/salt aqueous two-phase systems;micellar aqueous two-phase systems;biological particles;virus;adenovirus
Most of the metals released from industrial activity, among them are cadmium (Cd) and nickel (Ni)... more Most of the metals released from industrial activity, among them are cadmium (Cd) and nickel (Ni), inhibit the productivity of cultures and affect microbial metabolism. In this context, the aim of this work was to investigate the capacity of sugar cane vinasse to mitigate the adverse effects of Cd and Ni on cell growth, viability, budding rate and trehalose content of Saccharomyces cerevisiae, likely because of adsorption and chelating action. For this purpose, the yeast was grown batch-wise in YED medium supplemented with selected amounts of vinasse and Cd or Ni. The negative effects of Cd and Ni on S. cerevisiae growth and the mitigating one of sugar cane vinasse were quantified by an exponential model. Without vinasse, the addition of increasing levels of Cd and Ni reduced the specific growth rate, whereas in its presence no reduction was observed. Consistently with the well-proved toxicity of both metals, cell viability and budding rate progressively decreased with increasing their concentration, but in the presence of vinasse the situation was remarkably improved. The trehalose content of S. cerevisiae cells followed the same qualitative behavior as cell viability, even though the negative effect of both metals on this parameter was stronger. These results demonstrate the ability of sugar cane vinasse to mitigate the toxic effects of Cd and Ni.
The effects of template surface composition on fluorinated surfactant segregation were investigat... more The effects of template surface composition on fluorinated surfactant segregation were investigated for imprint lithography with photopolymerizable vinyl ether formulations. Heptadecafluoro-1,1,2,2-tetrahydrodecyl vinyloxy-methyloxy dimethylsilane, containing a vinyl ether group, was employed as the surfactant, and blanket templates were pressed onto the liquid and illuminated with UV radiation from below. The extent of surfactant segregation to the vinyl ether-template interface before polymerization was characterized using contact angle measurements and angle-resolved X-ray photoelectron spectroscopy after removing the template from the cured vinyl ether polymer. Blanket surfaces consisting of bare quartz, high-density polyethylene, and quartz treated with tridecafluoro-1,1,2,2,-tetrahydrooctyltrichlorosilane afforded templates with different surface energy and polarity. The highest degree of surfactant segregation was found with tridecafluoro-1,1,2,2,-tetrahydrooctyltrichlorosilane-treated quartz, whereas little surfactant segregation was found for bare quartz. A thermodynamic model is developed to predict the surface segregation profiles.
The thermal stability of the recombinant green fluorescent protein (GFPuv) expressed by Escherich... more The thermal stability of the recombinant green fluorescent protein (GFPuv) expressed by Escherichia coli cells and isolated by three-phase partitioning extraction with hydrophobic interaction chromatography was studied. The GFPuv (3.5–9.0 μg of GFPuv/mL) was exposed to various pH conditions (4.91–9.03) and temperatures (75–95°C) in the 10 mM buffers: acetate (pH 5.0–7.0), phosphate (pH 5.5–8.0), and Tris-HCl (pH 7.0–9.0). The extent of protein denaturation (loss of fluorescence intensity) was expressed in decimal reduction time (D-value), the time exposure required to reduce 90% of the initial fluorescence intensity of GFPuv. For pH 7.0 to 8.0, the thermostability of GFPuv was slightly greater in phosphate buffer than in Tris-HCl. At 85°C, the D-values (pH 7.1–7.5) ranged from 7.24 (Tris-HCl) to 13.88 min (phosphate) The stability of GFPuv in Tris-HCl (pH>8.0) was constant at 90 and 95°C, and the D-values were 7.93 (pH 8.38–8.92) and 6.0 min (pH 8.05–8.97), respectively. The thermostability of GFPuv provides the basis for its potential utility as a fluorescent biologic indicator to assay the efficacy of moist-heat treatments at temperatures lower than 100°C.
To determine the influence of various culture conditions, transformed cells of Escherichia coli e... more To determine the influence of various culture conditions, transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) were grown in nine cultures with four variable conditions (storage of inoculated broth at 4°C prior to incubation, agitation speed, isopropyl-β-d-thiogalactopyranoside [IPTG] concentration, and induction time). The pelleted cells were resuspended in extraction buffer and subjected to the three-phase partitioning (TPP) extraction method. To determine the most appropriate purification resin, protein extracts were eluted through one of four types of HiTrap hydrophobic interaction chromatography (HIC) columns prepacked with methyl, butyl, octyl, or phenyl resins and analyzed further on a 12% sodium dodecylsulfatepolyacrylamidegel. With Coomassie staining, a single band between 27 (standard GFPuv) and 29 kDa (molecular weight standard) was visualized for every HIC column sample. TPP extraction with HIC elution provided about 90% of the GFPuv recovered and eight-fold GFPuv enrichment related to the specific mass. Rotary speed and IPTG concentration showed, respectively, greater negative and positive influences on GFPuv expression at the beginning of the logarithmic phase for the set culture conditions (37°C, 24-h incubation).
Revista Brasileira De Ciencias Farmaceuticas, 2007
In this work, the effect of different parameters for extraction of casein and bovine serum albumi... more In this work, the effect of different parameters for extraction of casein and bovine serum albumin (BSA) were studied. Such proteins were extracted by soybean lecithin reversed micelles. BSA extraction was not effective, independent of the extraction conditions employed. Owing to its molar mass, the effect of exclusion by size was clearly observed. The casein extraction yield increased about 23-fold as a function of agitation time. In other words, the increase occurred by using higher contact time between protein and reversed micelles. The use of hexanol as a co-solvent was effective, and increased casein extraction to 36%. The extraction values were strongly influenced by pH, and the high extraction yield was obtained under the following conditions: 20% at pH 7.9, 80% at pH 5.4 e 100% at pH 5.0 (close to casein isoelectrical point).
Transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) we... more Transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) were subjected to two methods of extraction: (1) freezing/thawing/sonication (FTS) cycles prior to the three-phase partitioning (TPP) method, or (2) directly to TPP extraction. The amount of GFPuv released by the FTS plus TPP method varied: 374µg/mL (first cycle), 93–442 µg/mL (second cycle), 32–359 µg/mL (third cycle), 18–115 µg/mL (fourth cycle). The GFPuv yields by the second method (TPP only) were, 23–54 µg/mL for the first extract and 33–91 µg/mL for the second. The FTS plus TPP method released similar amounts of GFPuv to that extracted by TPP; and provided a better mixture elution through the hydrophobic interaction column: 13–63 µg/mL for FTS plus TPP methods, and 2.5–13 µg/mL for TPP. The results showed that although selective permeation is a more laborious methodology, it was more efficient for obtaining of GFPuv in relation to the direct extraction of the cells for TPP.
Brazilian Archives of Biology and Technology, 2003
From analytical to commercial scale, aqueous two-phase systems have their application in the puri... more From analytical to commercial scale, aqueous two-phase systems have their application in the purification, characterization and study of biomaterials. In order to improve the selectivity of the systems, the biospecific affinity ligands were introduced. In the affinity partitioning aqueous two-phase system, have many enzymes been purified. This review discusses the partitioning of some enzymes in the affinity aqueous two-phase systems in regard to the different ligands, including reactive dyes, metal ions and other ligands. Some integration of aqueous two-phase system with other techniques for more effective purification of enzymes are also presented.
Bioprocess, Bioseparation, and Cell Technology, 2009
ABSTRACT The study of biomolecules and bioparticles has achieved great advances, thus increasing ... more ABSTRACT The study of biomolecules and bioparticles has achieved great advances, thus increasing the productivity with upstream improvements. Nowadays, downstream processes are the bottleneck in the production of some biopharmaceuticals, a change from the previous years' scene. Current purification platforms will reach their physical limits at some point, creating the necessity for new approaches. This article reviews an alternative method for the extraction and purification of biomolecules/bioparticles denominated as aqueous two-phase systems (ATPSs). Biocompatibility and readiness to scale up are some of the characteristics of ATPSs and we also discuss its application in the biotechnology field.Keywords:polymer/polymer aqueous two-phase systems;polymer/salt aqueous two-phase systems;micellar aqueous two-phase systems;biological particles;virus;adenovirus
Most of the metals released from industrial activity, among them are cadmium (Cd) and nickel (Ni)... more Most of the metals released from industrial activity, among them are cadmium (Cd) and nickel (Ni), inhibit the productivity of cultures and affect microbial metabolism. In this context, the aim of this work was to investigate the capacity of sugar cane vinasse to mitigate the adverse effects of Cd and Ni on cell growth, viability, budding rate and trehalose content of Saccharomyces cerevisiae, likely because of adsorption and chelating action. For this purpose, the yeast was grown batch-wise in YED medium supplemented with selected amounts of vinasse and Cd or Ni. The negative effects of Cd and Ni on S. cerevisiae growth and the mitigating one of sugar cane vinasse were quantified by an exponential model. Without vinasse, the addition of increasing levels of Cd and Ni reduced the specific growth rate, whereas in its presence no reduction was observed. Consistently with the well-proved toxicity of both metals, cell viability and budding rate progressively decreased with increasing their concentration, but in the presence of vinasse the situation was remarkably improved. The trehalose content of S. cerevisiae cells followed the same qualitative behavior as cell viability, even though the negative effect of both metals on this parameter was stronger. These results demonstrate the ability of sugar cane vinasse to mitigate the toxic effects of Cd and Ni.
The effects of template surface composition on fluorinated surfactant segregation were investigat... more The effects of template surface composition on fluorinated surfactant segregation were investigated for imprint lithography with photopolymerizable vinyl ether formulations. Heptadecafluoro-1,1,2,2-tetrahydrodecyl vinyloxy-methyloxy dimethylsilane, containing a vinyl ether group, was employed as the surfactant, and blanket templates were pressed onto the liquid and illuminated with UV radiation from below. The extent of surfactant segregation to the vinyl ether-template interface before polymerization was characterized using contact angle measurements and angle-resolved X-ray photoelectron spectroscopy after removing the template from the cured vinyl ether polymer. Blanket surfaces consisting of bare quartz, high-density polyethylene, and quartz treated with tridecafluoro-1,1,2,2,-tetrahydrooctyltrichlorosilane afforded templates with different surface energy and polarity. The highest degree of surfactant segregation was found with tridecafluoro-1,1,2,2,-tetrahydrooctyltrichlorosilane-treated quartz, whereas little surfactant segregation was found for bare quartz. A thermodynamic model is developed to predict the surface segregation profiles.
The thermal stability of the recombinant green fluorescent protein (GFPuv) expressed by Escherich... more The thermal stability of the recombinant green fluorescent protein (GFPuv) expressed by Escherichia coli cells and isolated by three-phase partitioning extraction with hydrophobic interaction chromatography was studied. The GFPuv (3.5–9.0 μg of GFPuv/mL) was exposed to various pH conditions (4.91–9.03) and temperatures (75–95°C) in the 10 mM buffers: acetate (pH 5.0–7.0), phosphate (pH 5.5–8.0), and Tris-HCl (pH 7.0–9.0). The extent of protein denaturation (loss of fluorescence intensity) was expressed in decimal reduction time (D-value), the time exposure required to reduce 90% of the initial fluorescence intensity of GFPuv. For pH 7.0 to 8.0, the thermostability of GFPuv was slightly greater in phosphate buffer than in Tris-HCl. At 85°C, the D-values (pH 7.1–7.5) ranged from 7.24 (Tris-HCl) to 13.88 min (phosphate) The stability of GFPuv in Tris-HCl (pH>8.0) was constant at 90 and 95°C, and the D-values were 7.93 (pH 8.38–8.92) and 6.0 min (pH 8.05–8.97), respectively. The thermostability of GFPuv provides the basis for its potential utility as a fluorescent biologic indicator to assay the efficacy of moist-heat treatments at temperatures lower than 100°C.
To determine the influence of various culture conditions, transformed cells of Escherichia coli e... more To determine the influence of various culture conditions, transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) were grown in nine cultures with four variable conditions (storage of inoculated broth at 4°C prior to incubation, agitation speed, isopropyl-β-d-thiogalactopyranoside [IPTG] concentration, and induction time). The pelleted cells were resuspended in extraction buffer and subjected to the three-phase partitioning (TPP) extraction method. To determine the most appropriate purification resin, protein extracts were eluted through one of four types of HiTrap hydrophobic interaction chromatography (HIC) columns prepacked with methyl, butyl, octyl, or phenyl resins and analyzed further on a 12% sodium dodecylsulfatepolyacrylamidegel. With Coomassie staining, a single band between 27 (standard GFPuv) and 29 kDa (molecular weight standard) was visualized for every HIC column sample. TPP extraction with HIC elution provided about 90% of the GFPuv recovered and eight-fold GFPuv enrichment related to the specific mass. Rotary speed and IPTG concentration showed, respectively, greater negative and positive influences on GFPuv expression at the beginning of the logarithmic phase for the set culture conditions (37°C, 24-h incubation).
Revista Brasileira De Ciencias Farmaceuticas, 2007
In this work, the effect of different parameters for extraction of casein and bovine serum albumi... more In this work, the effect of different parameters for extraction of casein and bovine serum albumin (BSA) were studied. Such proteins were extracted by soybean lecithin reversed micelles. BSA extraction was not effective, independent of the extraction conditions employed. Owing to its molar mass, the effect of exclusion by size was clearly observed. The casein extraction yield increased about 23-fold as a function of agitation time. In other words, the increase occurred by using higher contact time between protein and reversed micelles. The use of hexanol as a co-solvent was effective, and increased casein extraction to 36%. The extraction values were strongly influenced by pH, and the high extraction yield was obtained under the following conditions: 20% at pH 7.9, 80% at pH 5.4 e 100% at pH 5.0 (close to casein isoelectrical point).
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